KR102100307B1 - Isolation and use of human lymphoid organ-derived suppressive stromal cells - Google Patents

Abstract

It provides a method for suppressing an immune response. The method comprises administering isolated lymphoid tissue-derived inhibitory stromal cells (LSSC) to an individual in need of treatment in an amount effective to suppress an immune response in the individual. In addition, the present invention relates to a method for isolating LSSC by digesting a lymphoid tissue fragment using a combination of enzyme mix and agitation, and then collecting LSSC. Also provided is a pharmaceutical composition comprising LSSC.

Description

Isolation and use of human lymphoid organ-derived inhibitory stromal cells {ISOLATION AND USE OF HUMAN LYMPHOID ORGAN-DERIVED SUPPRESSIVE STROMAL CELLS}

This application is made in US Patent Law 35 U.S.C. Priority is claimed on US Provisional Application No. 61 / 613,697 of March 21, 2012 based on § 119 (e), the contents of which are incorporated herein by reference in their entirety.

The present invention was carried out with government support by NIH grants DK074500, K01DK087770 and AI045757. Accordingly, the government has certain rights to the invention.

Immunosuppressants are used to inhibit or prevent the activity of the immune system to treat certain diseases. Clinically, immunosuppressants treat or prevent a number of symptoms, such as preventing rejection of transplanted organs and tissues (eg, bone marrow, heart, kidneys, liver, etc.), rheumatoid arthritis, multiple sclerosis, psoriasis and inflammatory It is used to treat inflammatory diseases such as bowel disease or autoimmune diseases. Immunosuppressive agents, which are currently approved for the treatment of chronic inflammatory diseases, should be administered frequently and carry serious side effects such as risk of kidney toxicity and onset.

Immunosuppressive stromal cell types (eg, mesenchymal stromal cells) have been initiated, but have not been fully characterized and did not meet the primary efficacy endpoint in clinical trials. In addition, separation of these stromal cells remains a challenge, due to the difficulty of successful separation of cells. Given the severity and vitality of conditions caused by unwanted immune activity, such as autoimmune or inflammatory diseases, there is a great need to develop effective treatments for these diseases.

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The present invention, in some aspects, relates to the discovery that stromal cells isolated from lymph nodes can inhibit the immune response. The ability of lymph node-derived stromal cells to inhibit an immune response in combination with its attack on known immune cells is an effective tool to suppress the immune response in an individual. Accordingly, some aspects of the present invention include a method of inhibiting an immune response by administering an isolated lymphoid tissue-derived inhibitory stromal cell (LSSC) to an individual in need of treatment in an amount effective to suppress the immune response in the individual. . In some embodiments, the LSSC administered to the individual is a cell amplified ex vivo. In some embodiments, the LSSC administered to the subject is substantially free of non-LSSC. In some embodiments, the LSSC is administered to the subject at least 100,000 LSSC / kg. In some embodiments, the LSSC is administered to the subject at least 1,000,000 LSSC / kg.

LSSC can be obtained from any one or combination of the following tissues: lymph nodes, spleen, thymus, tonsils, adenoids, and Peyer's patch. LSSCs can be autologous, allogeneic or heterologous tissue of an individual.

In some implementations, the LSSC resides on or within a two-dimensional or three-dimensional framework implanted in an individual. In some embodiments, LSSC is administered to a subject by intravenous, intraperitoneal, intraarterial, subcutaneous, or intramuscular injection, or by topical administration to a lesion, organ, organ capsule, fat or lymph node. do.

An individual in need of treatment refers to an individual with autoimmune disease or inflammatory disease, for example. Examples of autoimmune diseases or inflammatory diseases include, but are not limited to, rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, graft versus host disease, and sepsis.

In one aspect of the invention, a method for isolating LSSCs is provided. The method includes digesting fragments of lymphoid tissue and collecting LSSC using a combination of an enzyme mix and agitation.

The breakdown of lymphatic tissue fragments,

(i) incubating the lymphoid tissue fragment with an enzyme mix;

(ii) stirring the tissue with a pipette, followed by incubation to precipitate large fragments;

(iii) removing the supernatant and performing it in a series of steps, including repeating steps (i) and (ii) until all fragments are resolved,

Here, LSSC is collected by combining the above supernatant fractions and centrifuging to obtain a cell pellet.

In an embodiment, the isolated LSSC is cultured in a culture medium including a basic cell culture medium to which one or more of growth factor, serum, platelet cell lysate and antibiotic is added. In an embodiment, the isolated LSSC can be cultured at a density of 1-10,000 cells / cm ² . In an embodiment, the isolated LSSC is cultured under a partial pressure of oxygen of 0.1-21%. In some embodiments, LSSC can be cultured until there is substantially no non-LSSC. In some embodiments, LSSC is cultured until the number of stromal cells increases by at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200% or more. In some embodiments, a population comprising LSSC is enriched by removing hematopoietic cells or other non-substrate cells, or by culturing the cell population under conditions favorable to proliferation of stromal cells relative to the proliferation of non-substrate cells. (enrichment). These conditions are described herein.

In one embodiment, the enzyme mix comprises culture medium, Dispase, collagenase and DNaseI. LSSC can be derived from lymph nodes, spleen, thymus, tonsils, pharyngeal tonsils, and / or Fayer's plate. In some embodiments, LSSCs isolated using the methods described herein can be used to inhibit an immune response in an individual.

In one aspect of the invention, a composition comprising isolated lymphoid tissue-derived inhibitory stromal cells (LSSC) is provided. The LSSC can be separated using the method described above.

In one aspect of the present invention, there is provided a pharmaceutical formulation comprising a composition of isolated lymphoid tissue derived inhibitory stromal cells (LSSC). LSSC can be isolated by processing lymphatic tissue fragments using one or more of chemical, mechanical and electrical cell separation processes and then collecting the LSSC.

In some embodiments, the isolated LSSC is amplified through cell culture. In some embodiments, the isolated LSSC is a cell amplified ex vivo. In some embodiments, the isolated LSSC is amplified by propagating the collected cells until there is substantially no non-LSSC present in the LSSC.

In some embodiments, the isolated LSSC co-expresses CD140a and PD-L2. In some implementations, the isolated LSSC co-expresses CD140a and LTBR. In some embodiments, isolated LSSCs co-express CD140a, PD-L2 and LTBR. In some embodiments, the isolated LSSC expresses one or more other lymphoid markers selected from the group consisting of PD-L1, Thy-1, MADCAM-1, MYH11, IL-7R or ITGA7. In some embodiments, the isolated LSSC expresses one or more factors selected from the group consisting of IL-6, CCL19, CCL21 or VEGF.

LSSCs can be isolated from species selected from the group consisting of humans, non-human primates, dogs, cats, horses, pigs, cows, and rodents. LSSC can inhibit the proliferation of T cells in vivo or in vitro.

Each embodiment and each aspect of the present invention can be performed independently or in combination. In addition, the expressions and terminology used herein are for the purpose of description only and are not to be considered limiting. The use of “including,” “comprising,” or “having,” “containing,” “concomitant,” and variations thereof, is meant to encompass the items listed hereafter and equivalents thereof as well as additional items.

These and other aspects of the invention, as well as various advantages and usefulness, will become apparent with reference to the detailed description. Each aspect of the invention can encompass various implementations as understood.

All documents disclosed in this application are incorporated herein by reference in their entirety.

1 schematically shows a process of decomposing and preparing murine lymph node stromal cells collected to classify cells. 1. Enzyme mix (RPMI-1640 + 0.8mg / ml dispase, 0.2mg / ml collagenase P, 0.1mg / ml DNase I) is added to the tissue and incubated in a 37 ° C water bath. 2. After 10-20 minutes (see method), the lymph node fragments are stirred using a 1 ml pipette, and then incubated until the fragments precipitate. 3. The supernatant containing the dissociated cells is taken out, centrifuged, and stored on ice. 4. Repeat steps 1-3 until all fragments are completely resolved (no more than 60 minutes). 5. All supernatant fractions are collected, centrifuged and filtered before counting. 6. Add anti-CD45 microbeads and incubate at 4 ° C. on a rotating wheel. 7. Using MACS or AutoMACS, CD45 + cells are depleted and counted. 8. Stained concentrated CD45-stromal cells were stained with antibody; Sort at high purity using a 100 μm tip at 20 pso.
2 shows validation of a low-mortality method for isolating lymph node stroma subspecies. 2A shows the verification results for single cell suspension prepared using a low-lethal enzymatic digestion method. n = 6, mean + standard deviation. FIG. 2B depicts a typical flow cytometry profile for lymph node stroma subspecies isolated immediately from skin-draining lymph nodes or mesenteric lymph nodes in individual mice. 2C shows lymph node stroma subspecies identified by phosphorus drilling in frozen fragments of murine lymph nodes. Top panel: T cell zones FRC (non-grape Plata + CD31 ^-), and HEV (high endothelial venule) (grape Playa non ^- CD31 ^{+ +} cubic form). Two examples of HEVs are indicated by arrows. Central panel portion LEC water (grape Plastic Nin ⁺ CD31 ⁺⁾ and the water portion macrovascular ^- shows (grape Plastic non CD31 ^+). Bottom panels: coat afferent ^{(Capsular afferent) LEC (Lyve1 +} MadCAM +), MRC ( subcapsular, Lyve1 ^- MadCAM ⁺⁾ and FDC (follicular, Lyve1 ^-, MadCAM ^+). Co-location overlays are shown in white on an enlarged view. Original magnification: 200x. 2D depicts digestion and human lymph node (non-mesenteric origin) staining for flow cytometry analysis. The viscosity plot is the result of four independent experiments n = 4.
3 shows that the lymph node exhibits a site-specific modification to the matrix composition. Lymph nodes of individual C57Bl / 6 mice were digested and stained for flow cytometry. 3A shows lymph node cellularity. 3B depicts the proportion of CD45 negative lymph node stroma subspecies. 3C depicts the number of lymph node stroma subspecies. 3D depicts MadCAM ⁺ cells as a percentage of total FRC or LEC. 3E shows the total number of MadCAM ⁺ FRC or LEC. Figure 3F depicts the cell fidelity of skin-draining lymph nodes isolated from Rag ^-/- or WT (B6) mice of the same age. FIG. 3G depicts the number of lymph node stroma from Rag ^{− / −} or WT mice of the same age. FIG. 3G depicts the number of lymph node stroma from Rag ^{− / −} or WT mice of the same age. 3H shows the ratio of MadCAM + FRCs (MRC) or LEC in SLN of Rag ^{− / −} or WT mice of the same age, and FIG. 3I shows their number. Charts are for mouse n = 5. 3J shows the mean fluorescence intensity (MFI) for MadCAM staining in MRC or MadCAM + LEC. WT = wild type. * P <0.05; ** P <0.01 Mann-Whitney U test. Mice n = 5-10 in 2-3 independent experiments.
Figure 4 shows the results of thickening and classification of lymph node stromal cells. In Figure 4A, skin-draining lymph nodes derived from 6-10 C57Bl / 6 mice were enzymatically digested, and then CD45 ^- stromal cells were enriched using autoMACS. The number of charged (input) and recovered (calculated) CD45 ^- stromal cells in the column was plotted. Data show independent experiment n = 5. In Figure 4B,% of stromal cell yield was calculated via autoMACS enrichment of the stromal. Figure 4C shows the flow cytometry profile of stromal cells before and after autoMACS enrichment of the stromal cells. FIG. 4 depicts the sorting strategy and purity after sorting for major subspecies of lymph node stromal cells. Dot plots are gated on CD45 ^- propidium iodide ^- substrate.
FIG. 5 depicts upregulation of site-specific transcription of cytokines in murine FRCs derived from skin-draining lymph nodes. 5A shows the results of microarray analysis comparing FRCs sorted from skin-draining lymph nodes (SLN) or mesenteric lymph nodes (MLN). The dot plot shows the P-value versus fold-change results of SLN versus MLN using 15,486 sort probes. Probes up-regulated in SLN are indicated in red, and probes up-regulated in MLN are indicated in blue (P <0.05 and fold change>2; t-test). Independent identical experiment n = 4-5. Figure 5B depicts genes enriched in cytokines and SLNs encoding cytokine receptors (KEGG pathway mmu04060; P <0.015;Fisher's modified accuracy test + Benjamini correction) ). It shows the fold-increase of mean expression in SLN versus MLN.
6 shows that the three-dimensional (3D) culture of muline FRC mimics in vivo function. In FIG. 6A, lymph nodes were disassembled, a single cell suspension was added to the culture for 5 days, then trypsinized and stained for flow cytometry profiling. Stromal cells are CD45 was identified using the (left panel) and CD31 following grape Plastic ^non-stained with (right panel, CD45 search gating in). In particular, CD45 is a marker for hematopoietic cells and is therefore not present on stromal cells. In Figure 6B, the cultured stromal cells were stained with FDC-M1 or the corresponding isotype control. Figure 6C shows the results of CD31 staining for cultured stromal cells recovered with a high trypsin or low trypsin protocol. Dot plots represent three independent experiments, n = 3. In Figure 6D, cultured stromal cells were recovered and then purified by autoMACS. FRC, LEC or a mixture of FRC and LEC was inoculated with 2D (plastic tissue culture plate) or 3D (matrigel and collagen gel), and cultured again for 3 days, followed by imaging. The original magnification is 10x. The photographs show 2 independent experiments, n = 2. In FIG. 6E, the FRC purified using F-actin and DAPI was stained in 2D or 3D to visualize the network. The photo shows three independent experiments, n = 3. (F) Purified FRC was inoculated in 96-well plates with or without PP2 in 3D culture. After 24-36 hours, gel shrinkage was quantified. The photo shows three independent experiments, n = 3. Bar graphs represent mean + standard deviation, P <0.05, t-test. In FIG. 6G, the purified FRC was inoculated in 3D culture with purified dendritic cells (DC) or B cells, and then images were continuously taken every 90 seconds. The top panel is a picture captured at the original magnification, 20 x. The sub-panel depicts the cartoons of the photographed stromal cells and associated mobile white blood cells. In Figure 6H, lymph nodes were disassembled, single cell suspensions were added to the culture for 5 days, then trypsinized and stained for flow cytometry profile analysis.
7 shows that the seal lymphoid tissue-derived suppressor cells inhibit the proliferation of activated allogeneic splenocytes. The unfractionated LSSC was cultured with freshly isolated human spleen cells derived from membrane-related donors. Splenocytes were labeled with CFSE, a fluorescent dye that dilutes when cells divide. T cells in the spleen population were activated using PHA and IL-2. The histogram shows T cells identified through expression of the T cell antigen receptor. Ratio refers to the ratio of splenocytes to stromal cells.
8 shows that human LSSC inhibits the proliferation of activated heterologous T cells. The unfractionated LSSC was incubated with freshly isolated mouse spleen cells. Splenocytes were labeled with CFSE, which is diluted during cell division. T cells were then activated with anti-CD3 and anti-CD28 antibodies. The histogram shows T cells identified through expression of the T cell antigen receptor. The ratio represents the ratio of splenocytes to stromal cells.
9 shows that LSSC inhibits T cell proliferation through a novel mechanism. In FIG. 9A, unfractionated human LSSC was cultured with splenocytes freshly isolated from wild type or IFNγ-/-mice. Splenocytes were labeled with CFSE, which is diluted during cell division. T cells were then activated with anti-CD3 and anti-CD28 antibodies. The histogram shows T cells identified through expression of the T cell antigen receptor. The ratio represents the ratio of splenocytes to stromal cells. In Figure 9B, PDPN ^+, PDPN ^-, and differential hoekhwa a human blood purified by freshly separated from the LSSC unrelated donor-derived T cells were cultured with the cells. T cells were labeled with CFSE, which is diluted during cell division. Then, T cells were activated with PHA and IL-2. The histogram shows T cells identified through expression of the T cell antigen receptor. The supernatant was collected and tested for the presence of nitrite, a conversion product of nitric oxide.
FIG. 10 shows that the two major sub-population groups of human lymphoid tissue-derived suppressor cells inhibit proliferation of activated allogeneic T cells, respectively. In FIG. 10A, two subgroups of LSSC were purified based on the expression or deficiency of glycoprotein-36 (also called PDPN). Both subsubgroups were non-endothelial (CD31 negative) and non-hematopoietic (CD45 negative). In FIG. 10B, PDPN ⁺ LSSC PDPN ^- LSSC or unfractionated LSSC (LSSC: T cells) was incubated with purified human blood-derived T cells freshly isolated from unrelated donors at a 1: 5 ratio. T cells were labeled with CFSE, which is diluted upon cell division. T cells were activated with PHA and IL-2. The histogram shows T cells identified through expression of the T cell antigen receptor. The ratio represents the ratio of splenocytes to stromal cells.
Figure 11 shows that human LSSC stops dividing allogeneic T cells before activation. PDPN + LSSC, PDPN- LSSC, or unfractionated LSSC were freshly isolated from unrelated donors and cultured with purified human blood-derived T cells. T cells were labeled with CFSE, which is diluted upon cell division. After activating T cells with PHA and IL-2, they were allowed to divide for 2 days, and then some T cells were inoculated onto LSSC to re-stimulate, or re-vaccinated to re-stimulate without LSSC or re-stimulated without re-stimulation. . The histogram shows T cells identified through expression of the T cell antigen receptor. Ratios represent the ratio of LSSC to T cells.
FIG. 12 shows that human LSSC significantly reduces initial mortality in mice with severe graft-versus-host disease. Allogeneic bone marrow transplantation was performed on the recipient mice (after split-dose lethal investigation, iv injection of total bone marrow). Severe GVHD was induced upon transplantation by co-injecting donor-derived splenocytes, including allergic T cells. Human LSSC 1 x 10 ⁶ pieces after inducing GVHD was injected into the peritoneum at some point, in the control mice to give a saline (vehicle). Viability was monitored.
13 shows the transcription characteristics of LSSC. Transcription characteristics of human LSSC were obtained using flow cytometry for surface markers and RT-PCR for secreted factors (FIG. 13A), and mRNA transcriptome was obtained using the Affymetrix HuGene ST1.0 gene array. (Fig. 13B).
14 shows the morphological features of human LSSC. Fibroblast-like morphology was observed in LSSC cultures cultured in α-MEM supplemented with fetal bovine serum. It often exhibited long processes, lamellopodia, filopodia, ruffled leading edges, and focal adhesion. Original magnification: 100x.
Figure 15 shows that the substrate of mouse lymph nodes amplified in vitro reduces the lethality of acute sepsis shock.
Figure 16 shows that when murine FRC is administered at the time of treatment, it prevents sepsis in two types of mouse models. In FIG. 16A, 3-6 week old young C57Bl6 / J mice were treated with a dose of LD100, LPS, a polysaccharide that forms part of the bacterial cell wall and causes a strong systemic immune response. Since there are no active infections in these models, these models are referred to as "sterile sepsis". Mice were treated with FRC or saline in saline 4 hours after administration of LPS to mice. The graph shows the percent survival over time. In FIG. 16B, aseptic sepsis was induced in highly aged mice (18-24 months old) as detailed in FIG. 16A, followed by treatment with FRC or saline after 4 hours. Viability was monitored. In FIG. 16C, in a young mouse, the intestine is pierced twice with a needle to perforate the fecal matter into the abdominal cavity, followed by cecal ligation and perforation (CLP) sepsis, establishing a severe infection that progresses to blood sepsis within 24 hours Did. This model mimics a burst appendix, a gunshot wound, or other (intestinal) trauma. Autologous FRC was administered 4 hours after CLP. In FIG. 16D, CLP sepsis was induced in young mice and antibiotics were treated. Mice were administered saline, allogeneic FRC or allogeneic MSC as specified 4 hours after CLP. MSC (mesenchymal stromal cells) was used as a cellular-negative control. MSC is an inhibitory stromal cell type, and some authors have demonstrated that their effect on sepsis is minimal or no when administered at the time of treatment with homeopathic (> 60 min after septic attack) (Nemeth et. al. 2010; Mei et al. 2010). The results of the present invention likewise showed that MSC administration as a whole was not beneficial for viability, but allogeneic FRC provided significant advantages for viability. In FIG. 16E, mice of FIG. 16D were analyzed 16 hours after CLP. The concentrations of major cytokines in blood plasma were measured and compared to mice receiving saline or allogeneic FRC and untreated (non-septic) mice. The dotted line represents the lower and upper limits of reliable detection.
17 shows that murine FRC prevents death from colitis. Colonitis was induced by injecting purified native T cells into immunodeficient mice. The mice developed T cell-mediated inflammation against unidentified antigens (occurring in human disease), resulting in significant weight loss, colon enlargement and death. It is a model of human chronic colitis or Crohn's disease. Allogeneic FRC was administered 2 weeks after treatment (at the time of treatment) after the signal of disease appeared in the mouse and the weight began to decrease significantly. Compared to the brine-treated control, FRC was injected twice weekly from 2 to 6 weeks. Viability was monitored.
18 shows that human LSSC secretes soluble cytokine receptors. Human LSSC was cultured in DMEM with BSA added, and no stimulation or addition of exogenous factors was performed. 23 hours after inoculation with the soluble cytokine receptor protein, the Luminex MAGPIX system was detected in the culture medium using the manufacturer's instructions. Soluble cytokine receptors can bind to free cytokines and prevent pro-inflammatory functions in vivo.
19 shows that the LSSC isolated from the lymph nodes and tonsils are similar in phenotype. Human LSSCs derived from lymph nodes or tonsils were evaluated by flow cytometry for surface expression of the discriminator. All cells are CD45 negative and CD31 negative.
FIG. 20 shows that LSSC isolated from lymph nodes (FIGS. 20A and 20B) and tonsils (FIGS. 20C and 20D) have similar levels of T cell inhibition through soluble factors. Allogeneic T cells derived from human blood were labeled with CFSE, which is diluted with each cell division. T cells were then activated with PHA and hIL-2 with or without hLSSC isolated from lymph nodes or tonsils. hLSSCs were placed in wells or physically separated from T cells with a 0.4 μm transwell membrane that passed through the soluble factor but not the cells. T cells: Shows the percentage of T cells that divided once, twice, three times, or not at all when incubated with and without transwell at various ratios of hLSSC. The percent inhibition due to soluble factors was calculated as T cells divided using transwells / T cells divided without transwells x 100.

The present invention, in one aspect, relates to isolated lymphoid tissue-derived inhibitory stromal cells (LSSC). The present invention describes methods of isolating and amplifying LSSC in vitro in a commercially available manner, new compositions comprising LSSC, and uses of LSSC, particularly therapeutic uses for inhibiting autologous, allogeneic and heterologous immune responses do.

The present inventors have found that lymph node-derived stromal cells can inhibit the activity of the immune system. In addition, lymphoid tissue-derived stromal cells attract immune cells, which is not a known feature of other immunosuppressive stromal cell populations. The attraction of lymphoid tissue-derived stromal cells to attract immune cells such as T cells, B cells, NK cells, etc., combined with its immunosuppressive function, makes them highly effective therapeutic tools for suppressing immune responses in individuals. LSSC can be used to inhibit the activity of T cells, B cells, NK cells, NKT cells, dendritic cells and macrophages, alone or in combination with other immunosuppressive drugs.

Lymph nodes are important secondary lymphoid organs located at the lymph node in the body, enabling efficient interaction between antigen presenting cells and native T and B cells, and promoting efficient initiation of immune responses (Warnock et al., 1998, Mebius , 2003, Katakai et al., 2004a, Katakai et al., 2004b, Sixt et al., 2005, Bajenoff et al., 2006, Junt et al., 2008). Lymph node stromal cells also play a role in maintaining native T cells and possibly appear to be underestimated (Link et al., 2007).

Lymph nodes can be isolated and the obtained cells can be cultured in culture. “Lymph tissue-derived inhibitory stromal cells” refers to all cell types present in lymphoid tissue that can adhere to a tissue culture dish and maintain for a period of time. The cultured cells can be a heterogeneous population, with fibroblast reticulocytes (FRC), follicular dendritic cells (FDC), lymphoid endothelial cells (LEC), vascular endothelial cells (BEC) and marginal reticulum cells (MRC). Likewise, it can consist of most of the cells in the lymph node. In addition, the cultured cells can be a homogeneous population of FRC, FDC, LEC, BEC and / or MRC or any selection combination. In some embodiments, LSSCs are isolated using the methods described herein and amplified in vitro. LSSC can be derived from skin or mesenteric lymph nodes, spleen, thymus, tonsils, pharyngeal tonsils and / or Faer's plate. LSSC has no or is depleted of hematopoietic LSSC. In some embodiments, the LSSC is derived from a species selected from the group consisting of humans, non-human primates, dogs, cats, horses, pigs, cows, and rodents.

Distinguished stromal subcellular cells build the structure of the lymph nodes and maintain each microniche. Fibroblast reticulocytes (FRCs) in the T cell region express chemokines CCL19 and CCL21, attracting T cells and dendritic cells (DC), creating a complex meshwork of collagen and dendritic cells (DC) from which these cells ride. Accumulate (Ansel et al., 2000, Luther et al., 2000, Katakai et al., 2004b, Bajenoff et al., 2006, Link et al., 2007, Woolf et al., 2007). The follicular dendritic cells (FDC), similar to FRC, but specialized for B cell attraction, are rare subspecies specialized for B cell attraction and antigen presentation. FDC is found only in the B cell follicle and is produced through CXCL13 secretion (Cyster et al., 2000). The lymph node envelope is basically an enlarged lymphatic vessel constructed from lymphatic endothelial cells (LEC), which transports the interstitial fluid to the lymph nodes and transfers it to the parenchymal tissue. It passes through the bulk and cortex, ultimately reaching efferent lymphatics, which further transport the lymph upstream (Mebius, 2003, Willard-Mack, 2006). Blood endothelial cells (BEC) build cortical vessels and capillaries, including high endothelial venules, specialized to attract native T cells from the bloodstream (Mebius, 2003, Willard-Mack, 2006).

Other subspecies of several species have also been reported in the literature, but have not been fully studied, and their lineages and major functions have not been identified (Fletcher et al., 2011). Translational reticulocytes (MRCs) are CXCL13-producing stromal subspecies that grow in the cortical side of the subcapsular curve (Katakai et al., 2008). Although these cells showed similarity to FRC, including expression of grapeplanin, their origin was not confirmed (Katakai et al., 2008). In the spleen, similar stromal subspecies are important for the transfer of immature B cells to the follicle (Ansel et al., 2000). The assumption is that MRC is a postnatal equivalent of lymphoid tissue organizer (Lto) subspecies responsible for lymph node organization (Coles et al., 2010) (Katakai et al., 2008). Indeed, it has been fully demonstrated that post-natal staphylococcal + splenic matrix can be regenerated through interaction with hematopoietic lymphoid tissue inducer cells (Scandella et al., 2008).

LSSC is used in accordance with the present invention to suppress the immune response. That is, aspects of the present invention include a method of inhibiting an immune response by administering isolated lymphoid tissue-derived inhibitory stromal cells (LSSC) to an individual in need of such treatment in an amount effective to suppress the immune response in the individual.

As used herein, “isolated” means separating a stromal cell population from another non-substrate cell population. Separation can be performed by any process that separates cells, such as propagation conditions, purification techniques such as the use of magnetic beads, cell sorting, or other similar techniques. In some embodiments, LSSC is isolated using the methods described herein.

As used herein, “inhibiting” means lowering the obvious signs of an immune response in terms of level, severity, range, or duration, such as decreased activation of T cells or B cells and decreased fluid-mediated and cell-mediated responses. do. “Inhibition”, for example, examines the rate or presence of T or B cell division in the presence or absence of LSSC, or the secretion of cytokines by immune cells in the presence or absence of LSSC, or the presence or absence of LSSC It can be measured by measuring antibody production under or by measuring CTL function in the presence or absence of LSSC. Likewise, suppression of the immune response can be measured through symptoms, such as by measuring inflammation reduction or reduction of other symptoms associated with the disease being treated. "Inhibition" of an immune response refers to a reduction in clinical or other practically important level or severity, or range or duration, rather than the necessity of complete denial or prevention of any signs of this immune response. In one embodiment, inhibition is measured by examining the rate of cell division of pre-activated allogeneic T cells in the presence or absence of LSSC. In one embodiment, inhibition is measured by examining the mortality rate in mice with or without transplantation host disease in the presence or absence of LSSC. In one embodiment, inhibition is measured by examining mortality due to acute sepsis shock in mice.

In some embodiments, the individual in need of such treatment has an autoimmune disease or inflammatory disease.

The term 'autoimmune disease or inflammatory disease' refers to a disease state and condition in which the patient's immune response targets the patient's own components and renders them inadequate, sometimes significantly debilitating. As used herein, 'autoimmune disease or inflammatory disease' is intended to further include autoimmune conditions, syndromes, and the like. Examples of autoimmune diseases or inflammatory diseases include, but are not limited to, rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, graft versus host disease, and sepsis.

In some embodiments, the individual in need of treatment is an individual identified as having one or more of the conditions described above, ie, the individual is suffering from the disease or condition described above (e.g., using methods well known in the art). ) It is an individual diagnosed by a doctor. In some embodiments, an individual in need of treatment is suspected of having or developing an ailment or condition as described above, such as an individual exhibiting one or more symptoms (e.g., severe fatigue or cartilage pain) indicative of the ailment or condition described above. Being an individual. In some embodiments, the individual in need of treatment is an individual who has one or more risk factors for developing an autoimmune disease or inflammatory disease. Risk factors include, but are not limited to, gender, age, genetic predisposition, autoimmune disease, or chronic inflammatory disease onset and lifestyle. The term “individual in need of treatment” also includes a person who has suffered from the above-mentioned disease or condition, and whose symptoms have been alleviated.

In some embodiments, the individual in need of treatment is an individual suffering from or suspected of having an autoimmune disease or an inflammatory disease. The subject may exhibit abnormal titers, for example, autoantibodies.

The individual is an animal, typically a mammal. In one aspect, the individual is a dog, cat, horse, sheep, goat, cow, or rodent. In major embodiments, the individual is a human.

LSSC is administered in an effective amount. An effective amount is an amount sufficient to provide a medically desirable result, and can be determined by those skilled in the art using conventional methods. In some embodiments, an effective amount is an amount that results in any improvement in the condition being treated. In some embodiments, an effective amount can depend on the type and level of the disease or condition being treated and / or on one or more additional therapeutic agents. However, one of ordinary skill in the art can determine the appropriate dosage and range of therapeutic agents to be used, for example, based on in vitro and / or in vivo testing and / or knowledge of other compound dosages.

When administered to a subject, an effective amount of therapeutic agent, as well as the individual disease being treated; Severity of the disease; It will depend on individual patient parameters such as age, physical condition, height and weight, combination treatment, frequency of treatment and mode of administration. These factors are well known to those skilled in the art and can be seen by routine experimentation. In some embodiments, the highest dose is used, ie, the highest safe dose according to appropriate medical judgment.

When treating an autoimmune disease or inflammatory disease, an effective amount is an amount that slows the progression of the disease, stops the progression of the disease, or reverses the progression of the disease. An effective amount includes an amount necessary to slow, slow, inhibit, alleviate or revert one or more symptoms associated with an autoimmune disease or inflammatory disease. In some embodiments, these terms refer to pain, fatigue and / or fever reduction or swelling of one or more joints associated with an autoimmune or inflammatory disease. In some embodiments, these terms refer to a decrease in the level of circulating autoantibodies associated with autoimmune diseases or inflammatory diseases. In some embodiments, these terms refer to a reduction in human PASI score. In some embodiments, these terms refer to an improvement in the human global assessment score.

In some embodiments, one or more doses of LSSC per kg of an individual are administered to the individual at a dose of one or more days (depending on the dosing mode course and factors described above) for several days. In some embodiments, the individual is administered at least 1 million LSSC per kg of individual. In some embodiments, at least 10 million LSSCs per kg of individual are administered to the individual. The actual dosage level of a therapeutic agent can be altered to obtain an amount effective to achieve the desired therapeutic response for the individual patient, composition and mode of administration. The dosage level chosen will depend on the activity of the individual compound, the route of administration, the tissue being treated and the history of treatment of the existing patient. However, within the knowledge of the art, it includes starting the administration of the compound at a level less than necessary to achieve the desired therapeutic result, gradually increasing the dosage until the desired effect is achieved.

In some embodiments, the LSSC administered to the individual is a cell amplified ex vivo. LSSC is amplified ex vivo by culturing cells in culture medium. The isolated LSSC may be cultured in a culture medium including a basic cell culture medium to which one or more of growth factors, serum, platelet cell blood, and antibiotics are added. In some embodiments, isolated LSSCs are cultured in minimal essential medium (MEM) α medium to which, for example, fetal bovine serum and penicillin / streptomycin have been added. In some embodiments, the LSSC administered to the subject is substantially free of non-LSSC. In some embodiments, the LSSC is autologous, homologous or heterologous to the individual.

Pharmaceutical preparations comprising LSSC are administered to an individual via any suitable route. For example, the composition can be administered to a subject by intravenous, intraperitoneal, intraarterial, subcutaneous or intramuscular injection, or by topical administration onto or into lesions, organs, organ capsules, fats or lymph nodes.

In some implementations, the LSSC is on or within a two-dimensional or three-dimensional framework implanted into an individual. Implants or matrices include polymer matrices, such as devices based on fibers or hydrogels. The implant may or may not be biodegradable. Fibrous or hydrogel implants can be made or constructed using commercially available materials. LSSC is inoculated onto the implant by applying a cell suspension to the implant. This can be achieved by immersing the implant in a cell culture container or by injecting the cell into the implant or by other direct application. Cell-implanted implants are implanted using standard surgical techniques. The implant can be inoculated and cultured in vitro prior to implantation, transplanted immediately upon inoculation, or after implantation and then inoculated with cells. In a preferred embodiment, the cells are inoculated onto or into the implant and cultured in vitro for about 10 hours to 2 weeks.

Some aspects of the invention include a method of isolating lymphoid tissue-derived inhibitory stromal cells (LSSC). The method comprises the steps of digesting lymphoid tissue fragments using a combination of enzyme mix and agitation and then collecting the LSSC. In some embodiments, the degradation of the lymphoid tissue fragment comprises (i) incubating the lymphoid tissue fragment with an enzyme mix; (ii) stirring the tissue using a pipette, followed by incubation to precipitate large fragments; (iii) The supernatant is taken out and carried out in a series of steps, including the steps of repeating steps (i) and (ii) until all fragments are degraded. Then, all the upper fractions were collected and centrifuged to obtain a cell pellet, whereby LSSC was collected. In some embodiments, the enzyme mix comprises culture medium, dispase, collagenase and DNase I. LSSC can be derived from skin or mesenteric lymph nodes, spleen, thymus, tonsils, pharyngeal tonsils and / or Faer's plate. The isolated cells can be used to suppress the immune response as described above.

The isolated LSSC may be cultured in a culture medium including a basic cell culture medium to which one or more of growth factors, serum, platelet cell blood, and antibiotics are added. In some embodiments, LSSC is cultured in minimal essential medium (MEM) α medium supplemented with fetal bovine serum and penicillin / streptomycin. In some embodiments, isolated LSSCs are cultured at an oxygen partial pressure of 0.1-21%. In some embodiments, isolated LSSCs are cultured at an oxygen partial pressure of 2.5-21%.

In some embodiments, the LSSC is cultured until there is substantially no non-LSSC present in the LSSC. In some embodiments, LSSC is cultured until the number of stromal cells increases by at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200% or more. In some embodiments, isolated LSSCs are cultured at a density of 1-10,000 cells / cm ² . In some embodiments, isolated LSSCs are cultured at a density of 10-500 cells / cm ² .

In some aspects of the invention, compositions are provided. The composition comprises isolated lymphoid tissue-derived inhibitory stromal cells (LSSC), where the LSSC is isolated by the methods described herein. For example, LSSC is separated by digesting lymphocyte tissue fragments using a combination of enzyme mix and agitation and then collecting the LSSC.

In some aspects of the invention, pharmaceutical formulations are provided. This pharmaceutical composition comprises a composition of isolated lymphoid tissue-derived inhibitory stromal cells (LSSC). LSSC can be isolated by processing lymphatic tissue fragments using one of chemical, mechanical and electrical cell separation processes and then collecting the LSSC. For example, cells can be separated by enzymatic or chemical degradation, physical destruction and agitation, and / or electric fields.

The isolated LSSC can be amplified through cell culture. In some embodiments, the isolated LSSC is an amplified cell ex vivo. In some embodiments, the isolated LSSC is amplified by culturing the collected cells until the LSSC is substantially free of non-LSSC. The cells can be cultured in a culture medium including a basic cell culture medium to which one or more of growth factors, serum, platelet cell blood, and antibiotics are added.

In some embodiments, isolated LSSCs co-express platelet-derived growth factor receptor, α polypeptide (PDGFRα or CD140a) and programmed cell death 1 ligand 2 (PD-L2 or CD273). In some embodiments, the isolated LSSC co-expresses CD140a and lymphotoxin beta receptor (LTBR). In some embodiments, isolated LSSCs co-express CD140a, PD-L2 and LTBR. In addition, the isolated LSSCs are programmed cell death 1 ligand 1 (PD-L1), Thy-1 cell surface antigen (Thy-1), mucosal vascular addressin cell adhesion molecule 1 (MADCAM-1) ), Myosin heavy chain 11 (MYH11), interleukin 7 receptor (IL-7R) or integrin α 7 (ITGA7). In some embodiments, the isolated LSSC is a group consisting of interleukin 6 (IL-6), chemokine (CC motif) ligand 19 (CCL19), chemokine (CC motif) ligand 21 (CCL21) or vascular endothelial growth factor (VEGF). Express one or more factors selected from.

LSSC can be isolated from species selected from the group consisting of humans, non-human primates, dogs, cats, horses, pigs, cows, and rodents, and inhibits T cell proliferation in vitro.

The pharmaceutical preparations of the present invention may include or be diluted with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” as used herein refers to one or more compatible fillers, diluents, or other materials suitable for administration to other mammals such as humans, dogs, cats or horses. The term "carrier" refers to organic or inorganic components, natural or synthetic substances, which combine active ingredients to facilitate utilization. The carrier can be mixed with the formulations of the present invention, and with each other, in a manner that does not have interactions that substantially impart desirable pharmaceutical efficacy or stability. Suitable carriers for oral, subcutaneous, intravenous, intramuscular, etc. formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

The invention is further illustrated by the following examples, but should not be construed as further limiting in any way. The entire contents of all references cited throughout this specification (references to documents, registered patents, published patent applications and pending patent applications) are expressly incorporated herein by reference.

Example

Materials and methods

mouse

Male C57Bl / 6 mice, 4-6 weeks old, were purchased from the Jackson Laboratory. The mice used in the ImmGen classification were age-matched at 6 weeks of age. Male C57BL / 6 Rag ^-/- mice were purchased from Taconic. All mice were allowed to rest for 5 days after transfer, and were kept under special sterility in accordance with the National Health Guidelines Association. Experimental procedures were conducted with the approval of the Research Animal Care Subcommittee at the Dana-Farber Cancer Institute.

Human lymph nodes

Human lymph nodes were obtained from donor carcasses through the National dieased Research Interchange (NDRI) Resource Center (Philadelphia, USA). Intact lymph nodes were transferred in DMEM on ice and processed for flow cytometry or treated for 24 hours of cell culture.

Antibody

For flow cytometry, the following antibodies were used for cell sorting of mouse lymph node stroma and staining of frozen fragments: anti-CD45 (clone 40-F11, BD Biosciences), anti-podoplanin (clone 8.1.1, Developmental Studies hybridoma Bank), anti-CD31 (clone MEC13.3, Biolegend), anti-Lyve 1 (clone 10.1.1, gifted from Dr. Andrew Farr) and anti-MadCAM (clone MECA-367, eBioscience). When it is appropriate to exclude dead cells and red blood cells, propidium iodide and clone TER119 (Biolegend) were used. The following antibodies are used for human cell staining: anti-CD45 (clone HI30, Biolegend), anti-CD31 (clone WM59, BD Biosciences) and anti-podoplanin (clone NZ-1, AngioBio Co), highly crossover -It was detected using adsorbed anti-rat IgG (H + L) Alexa-488 (Invitrogen).

Enzymatic digestion of individual mouse-derived lymph nodes

For flow cytometry analysis or cell culture, lymph nodes from individual mice were cut, drilled once using micro forceps and placed on ice in 50 ml of RPMI-1640. When the use of skin-draining lymph nodes was indicated, the axilla, brachial and inguinal lymph nodes were removed. After all lymph nodes were dissected, RPMI-1640 was removed, and RPMI with 0.8 mg / ml dispase and 0.2 mg / ml collagenase P (both purchased from Roche) and 0.1 mg / ml DNase I (Invitrogen) It was replaced with 2 ml of the prepared enzyme mix. The tubes were incubated at 37 ° C. in a water bath and slowly turned over at 5 minute intervals to allow the contents to mix well. After 20 minutes, the lymph nodes were carefully inhaled and released using a 1 ml pipette to break the capsule and release most of the white blood cells. The mixture was returned to the water bath, and the large fragments were allowed to settle for 30 seconds, after which the enzyme mix was removed, 10 ml of ice-cold FACS buffer (2% FCS, 5 mM EDTA / PBS) was added, followed by centrifugation (300 g, 4 minutes, 4 ° C). 2 ml of the fresh enzyme mix was placed in a digestion tube, the contents were carefully mixed with a 1 ml pipette, and then incubated while slowly mixing regularly using a 1 ml pipette. After 10 minutes, cells were mixed vigorously for 30 seconds using a 1 ml pipette. The fragments were precipitated again, the supernatant was transferred, placed in a cell pellet that had been previously spin down, and 2 ml of fresh enzyme mix was added to the digestion tube. Here, the digestion mix was mixed vigorously every 5 minutes using a 1 ml pipette until all residual lymph node fragments were completely resolved and cleared when illuminated with light. The process from the time of the first incubation to completion of the decomposition usually takes 50 minutes and never exceeds 60 minutes. The supernatant is centrifuged (300 g, 4 min, 4 ° C.) after each recovery, and finally the entire cell contents of the recovered lymph nodes of the individual mice are placed in each collection tube. Cells were filtered through an 80 μm nylon mesh, counted with a hemocytometer, and viability was assessed using trypan blue. Viability exceeded 95%. Then, 5 x 10 ⁶ cells per stain were incubated in 50 μl of diluted antibody and ice-cold FACS buffer (2% FCS, 5 mM EDTA / PBS) at 4 ° C. for 20 minutes, followed by FACSCalibur or FACSAria IIu (BD Biosciences). Captured.

Enzymatic digestion and matrix enrichment of mouse pool lymph nodes for cell sorting and large-scale separation

Disintegration of multiple mouse pools was performed overall as described in 2.1, with the following modifications (schematically depicted in FIG. 1): Lymph nodes obtained from 4-10 mice collected in one tube Next, it was digested using 5 ml of enzyme mix per digestion step. Lymph nodes were completely digested, cells were centrifuged and counted, and non-hematopoietic stromal cells were concentrated in a single cell preparation using a CD45 microbead and an autoMACS system (both purchased from Miltenyi). Incubated in FACS buffer at 4 ° C. on a rotating wheel for 20 minutes using ⁷ μl beads per ⁷ cells. The labeled fractions were depleted using the Depletes program according to the manufacturer's instructions. Then, it was stained using the antibody of the substantial volume for the classification coefficient was concentrated substrate, and cells (10 ⁶ cells diluted antibody cocktail 100㎕ per dog).

Enzymatic breakdown of human lymph nodes

Fat and connective tissue were carefully removed from the human lymph nodes, and then cut into small pieces (<0.5 cm) using fine scissors. The digestion was carried out according to section 2.4, and the enzyme concentration in RPMI-1640 was increased to 2.4 mg / ml dispase, 0.6 mg / ml collagenase P (both manufactured by Roche), and 0.3 mg / ml DNase I (Invitrogen). . If the entire organ could not be broken down, about 20 hybrid fragments representing the entire lymph node area were instead randomly selected. After 60 minutes, the lymph node fragment was completely degraded and, as described above, it was filtered, counted and stained as needed.

Classification of stromal cells by low pressure flow cytometry

Classification using flow cytometry of stromal cells is improved when using a special low pressure, large aperture setup, but is an innate technical problem in successfully classifying pure populations after resetting the classifier to non-standard items. There is. In the present invention, FACSDiva software was used and a FACSAria or upgraded FACSAria IIu device set to 20 psi and equipped with a 100 mm tip (all manufactured by BD Bioscience) was used. When each device was changed, the following approximate specifications were applied to four or more FACSAria devices set for substrate classification. These are different from the recommended setups specified by the manufacturer.

Reducing the stream pressure increases the size of the droplets. At 20 psi, only 5-6 droplets can be seen through the stream visualization window, but at 70 psi, 10-12 droplets can be seen. The breakoff point of the droplet directly affects the success of the classification and must be visible at all times, and the satellite resolution point may occur below the low pressure classification even after increasing the number of droplet occurrences. This makes successful sorting impossible, and it is important to be certain that satellite dissociation will result in 1-2 droplets below the screen. This can be reasonably established in two ways: Firstly, by temporarily adjusting the droplet breakoff point out of range higher than the view, the satellite dissociation point becomes visible, and the breakoff between the breakoff and the dissociation before re-adjusting downward. It can be established by counting the number of droplets and, secondly, estimating the number of droplets until dissociation occurs while looking at the shortening of the distance between the satellite and the droplet. Satellites, if not separated on-screen, should be close to separation.

Crucially, deciding whether or not to apply attenuation control that slows down the droplet amplitude will significantly change subsequent details. Records of sample sepup classifications with and without attenuation are provided for reference (Table 1). The BD Biosciences manual recommends attenuation that does not apply when sorted at 20 psi, but it has been found in the present invention that it is often necessary to use FACSAria (not FACSAria IIu) to generate a good stream. Manual adjustment of the 100 μm tip was necessary to generate a good stream using the FACSAria, but the FACSAria IIu system was not required to move the tip or apply damping if the o-ring was permanently mounted.

Finally, it was noted that the 633nm laser delay could change suddenly when combined with low pressure. The reasons for this are not clear, but we recommend setting the delay using CST or testing the laser function with SPHERO Rainbow Particles (BD Bioscience) (or any similar calibration check). It should be noted that the lack of signal from the 633nm laser means that laser delay may need to be re-corrected.

Compensation for stromal cell sorting should be done strictly using enriched stromal cells. The stroma is a cell that is large enough to carry autofluorescence. Devices supplemented with leukocytes dissociate populations suboptimally when classifying substrates. Since substrates are always rare in non-enriched populations, enriched substrates should be used for complementation, and in the present invention, autocalibration software is able to distinguish true autofluorescence levels and true substrate staining in dissociated single cell suspensions. There was no confirmation.

When the device is best calibrated, classification proceeds with excellent resolution and good efficacy of the classification stream. The classification stream as well as the bright individual points must be dissociated in the classification window; The "spraying" of the classification stream should not appear.

AutoMACS-enriched substrates were routinely classified as TRIzol (Invitrogen) with high purity (> 95%). ^{CD45-propidium iodide-after} gating on the substrate, the sub-species was classified as: fibroblast reticular cells (FRC), grape Plastic Nin ⁺ CD31 ^-; Lymphatic endothelial cell (LEC), non-grape Plata ⁺ CD31 ^-; Vascular endothelial cells (BEC), staphylanin ^- CD31 ⁺ ; Dual Voice stromal cells (DN), Ninh grape Plata ^- CD31 ^-.

Microarray analysis and analysis

RNA was isolated from previously sorted cells 10,000-15,000 weeks as previously mentioned (Yamagata et al., 2004) and amplified to generate Affymetrix GeneChip Mouse Gene using GeneChip Whole Transcript Sense Target Labeling Assay according to the manufacturer's instructions. 1.0 ST Arrays (Santa Clara, CA, USA). Thus, full-transcript information was obtained using probes designed to cross-link each gene at multiple locations. Raw data was normalized using the RMA (robust multi-array average) preprocessing algorithm (which performs background adjustment, quantile normalization and summarization) in the Expression File Creator module of GenePattern (Irizarry et al., 2003). Expression in one or more subspecies of the probe list (except when the mean expression value is <120 in all subspecies) and low variability between replicas (except when the coefficient of variation is> 0.5 in each population) Analysis). GenePattern's multiplot module was used to calculate the difference between skin lymph node FRC and mesenteric lymph node FRC. When the expression fold change was> 2 and P <0.05 (student's T test), differences were considered for further analysis. Output data is sorted using Microsoft Excel and analysis of enrichment in the KEGG pathway using the DAVID program's functional annotation tool (NIAID, david.abcc.ncifcrf.gov (Dennis et al., 2003)) in the gene list. Affymetrix exon background was set on the MoGene-1_0-st-v-1 chip. Results of KEGG pathway analysis with T-test P-values <0.05 after multiple hypothesis (benzamini) corrections were considered significant. The NCBI GEO listing number for the data series is GSE15907.

Lymph node stromal cell culture technique

Lymph nodes were dissociated, counted and inoculated at a concentration of 5 x 10 ⁵ cells / cm ² as described above. The cell culture medium was αMEM with 10% batch-tested low Ig FCS and 1% penicillin / streptomycin added. Plates were washed after 24 hours to remove non-adherent cells. Five days later, the mouse cell culture contained mainly LEC and FRC. Human cell cultures proliferated more slowly and were recovered after 7 days. Human lymph node stromal cell cultures mainly contained FRC and DN. To minimize the significant cell surface markers being cut by trypsin, a non-standard recovery protocol, consisting of a short incubation step in low concentration of trypsin with EDTA added, was commonly used (Table 2). Cells were washed with PBS to remove residual protein, and recovery buffer (0.2% trypsin in PBS + 5 mM EDTA) was added to the culture plate. Cells were incubated for 2 min at 37 ° C in recovery buffer. At this point, its shape was rounded, and complete medium was immediately added to the wells in the same volume. The cells of the plate were rinsed with gentle mixing using a 1 ml pipette, and the supernatant was added to another equal volume of complete medium for centrifugation (300 g, 3 min, 4 ° C.). FRC and LEC were reinoculated, sorted or purified as MACS from this formulation as needed.

For three-dimensional culture and network analysis, cells were prepared in the laboratory with high concentrations of rat tail collagen I (BD Biosciences), 1.8 mg / ml Matrigel basement membrane matrix (BD Bioscience), αMEM powder (Invitrogen) and 8.3% (v / v) Incubated in a deformable matrix, constructed with 5x αMEM stock medium and 30% (v / v) culture medium containing seed cells. The gel components were mixed on ice and inoculated with 200 μl of plastic or glass-bottom culture dish (MatTek) for imaging. The gel was fixed at 37 ° C for 10 minutes and then coated with a culture medium. For shrinkage analysis, gels were formed in a flat-bottomed 96-well culture plate and photographed 15 hours later. The shrinkage was measured by the following equation: Shrinked gel area (cm ² ) / well area (cm ² ) * 100 =% shrinkage. To photograph live cells by time-lapse, bone marrow-derived dendritic cells or B cells were mixed with purified FRC culture at a ratio of 5: 1. During imaging, the gel was incubated at 10% CO ₂ and 37 ° C. in αMEM with 10% FCS. The movie represents a time-lapse image with 90 seconds as one frame.

Immunohistochemistry and confocal microscopy

Lymph nodes were freshly harvested, fixed in 10% paraformaldehyde for 4 hours, and then placed in a 10% sucrose solution overnight. The blocks were then embedded in OCT compound (VWR), snap-frozen and stored at -80 ° C. Lymph nodes were cut into frozen fragments (7 μm) using a Leica cryostat, acetone fixed, and then blocked for 30 minutes using 2% BSA. After washing with PBS, the fragments were stained with primary antibody for 60 minutes, then washed, and then stained with secondary antibody for 60 minutes. The slide was covered with a cover slip using a fluorescent mounting medium (DAKO) and stored at 4 ° C. Images were captured using a Leica Sp5 confocal microscope.

Statistical analysis

Using GenePattern's multiplot program, T-test P values were calculated from normalized array data with coefficients of variation <0.5 between replicas for any sample. When the observed fold-change was also <2, the P-value <0.05 was considered significant. Genes were examined using the DAVID program (david.abcc.ncifcrf.gov) for KEGG pathway relevance, and data was compared using a T-test linked to a Benjamin Fisher's multi-theory calibration with a modified Fisher's accuracy test, Concentration of two or more genes was identified in a specific list. Flow cytometric data were compared using a non-parametric Mann-Whitney U test (Prism).

Isolation and culture of lymph node stromal cells

3-6 week old C57Bl6 / J mice were used as FRC donors. Lymph nodes of skin and mesentery were removed and dissociated using enzymatic and mechanical means. Lymph nodes obtained from 10 mice were regularly stirred at 37 ° C. for 60 minutes in RPMI-1640 with 0.8 mg / ml dispase, 0.2 mg / ml collagenase P and 0.1 mg / ml DNase added, and the enzyme mixture It was incubated with replacement. The decomposed cells were recovered by centrifugation. When digestion was complete, the cells were resuspended in RPMI-1640, filtered through an 80 mm mesh, and counted using a hemocytometer and trypan blue. Cells were seeded in culture flasks at 5 x 10 ⁵ cells / cm ² . The culture medium consisted of 10% batch-tested low Ig FCS in αMEM and 1% penicillin / streptomycin. The cells were washed after 24 hours, incubated again for 6 days, and then subcultured once using 0.2% trypsin and 5 mM EDTA in PBS, and then divided into 1:10 for amplification for 5-6 days again before use.

MSC isolation and culture

MSCs were recovered from the bone marrow of FRC donor mice described above. The femur and tibia were removed from 10 mice. This was centrifuged and re-suspended in sterile RPMI-1640 using a pipette to recover bone marrow. Cells were seeded in culture flasks at 5 x 10 ⁵ cells / cm ² . The culture medium consisted of 10% batch-tested low Ig FCS in αMEM and 1% penicillin / streptomycin. The cells were washed after 24 hours, incubated again for 6 days, then subcultured once using 0.2% trypsin and 5 mM EDTA in PBS, and then divided into 1:10 for amplification for 5-6 days again before use. .

Purification of FRC and MSC

Cells were recovered using 0.2% trypsin and 5 mM EDTA in PBS, then counted using a hemocytometer and trypan blue (survival> 95%), and anti-CD31-biotin 10 μl per cell 10 ⁷ -Suspended in 1 ml of FACS buffer (PBS + 2% FCS + 5 mM EDTA) to which 10 μl of CD45-biotin was added. After 15 minutes, cells were washed, incubated with anti-biotin microbeads, and then passed through a MACS LC column according to the manufacturer's instructions (Miltenyi Biotec). FRC purified by MACS was used in all experiments.

Sepsis induction: LPS model

C57Bl6 / J mice aged 3-6 weeks (young) or 18-24 months (aged) were injected once with LPS to generate aseptic septic. LPS 300 mg was dissolved in 100 μl of saline and administered by ip to young mice, and 150 mg of LPS was dissolved in 100 μl of saline and administered by ip. FRC 1 × 10 ⁶ was administered ip in a single injection at designated times in saline. Mice were euthanized when they reached any of the following endpoints: loss of consciousness, awakening, inability to stand up, or continued decrease in respiratory rate to less than 20 breaths per 10 seconds.

Sepsis induction: CLP model

Balb / c mice were subjected to appendectomy and perforation (CLP) to develop complex bacterial sepsis. Briefly, mice were anesthetized and incised in the midline to the skin and abdominal muscles. The location of the appendix was identified, taken out into a sterile surgical field, and 80% of the appendix was ligated with a 6.0 suture and a 2 puncture hole was made using a 23g needle. Several drops of fecal matter were discharged before the appendix was in place and sutured muscles and skin. The mouse was administered with 1 ml of saline as s, c ,. At the designated time points, mice were injected with saline or allogeneic FRC 1 × 10 ⁶ in saline once ip. At this time, all mice were treated with antibiotics (15 mg of ampicillin in saline was injected with ip). Antibiotics were administered every 12 hours. Mice were euthanized when one of the following endpoints was reached: loss of consciousness, awakening, inability to stand up, or persistent respiratory rate reduction of less than 20 breaths per 10 seconds.

Cytokine array

Blood was obtained as described above and plasma was collected by centrifugation. The concentration of the analyte was obtained using a Luminex MAGPIX array and a reader using xPONENT software according to the manufacturer's instructions.

Induction of colitis model

Eight week old female C / B17.scid mice were used as colitis recipients. A 7 week old Balb / c female donor mouse was used as a cell donor to induce colitis. Spleen, skin and mesenteric lymph nodes were extracted from 20 Balb / c mice and suspended in RPMI-1640 by mechanical disruption. Cells were filtered, counted and native CD4 + T cells were enriched using the CD4 ⁺ CD62L ⁺ CD44 ^low assay kit (R & D Systems) according to the manufacturer's instructions. Then, CD4 ⁺ CD45RB ^{hi +} T cells were sorted using FACS and resuspended in sterile RPMI-1640. 15 C / B17.scid mice were administered with 5 x 10 ⁵ cells in 100 μl of RPMI-1640. Mice started to lose weight 2 weeks after induction of colitis, and disease onset was confirmed. Then, during the duration of the experiment, mice were administered ip 1 x 10 ⁶ homogeneous FRCs once a week. Mice were euthanized upon reaching any of the following endpoints:> 20% reduction from starting weight or hind limb paralysis.

result

Enzymatic separation of mouse and human lymphoid stromal cells

Studies on lymph node stromal cells have recently been fueled, and some highly influential literature maintains native T cells (Link et al., 2007) and depletion (Lee et al., 2007, Nichols et al., 2007, Gardner et al., 2008, Magnusson et al., 2008, Yip et al., 2009, Cohen et al., 2010, Fletcher et al., 2010).

Accordingly, in the present invention, a low-lethal enzymatic digestion protocol was developed to enable highly reproducible isolation of low variability lymph node stromal cells. Using a combination of enzymatic dissociation and physical dissociation (Figure 1), immediately after separation, stromal cells were typically isolated with> 95% viability using trypan blue on the entire lymph node suspension (1.5% substrate) ( 2A). Six hours after separation and substrate thickening, the average viability of the substrate when using propidium iodide was 87.8% ± 0.8 (mean ± standard deviation, experiment n = 3). This high viability characterizes each substrate subspecies present in individual mice and makes their numbers comparable, which is a key factor in the successful isolation and culture of these cells. In the present invention, CD45, staphylanin, CD31 and MadCAM (FIG. 2B) were used to study five major subspecies of lymph node stroma. In particular, MadCAM + MRC subspecies have been isolated from flow cytometry analysis.

First, the strategy using flow cytometry of the present invention was directly compared with the in situ histological analysis of previously defined stromal subspecies (FIG. 2C). The aim of the present invention was to establish a method capable of monitoring changes in the structure of the substrate in parallel with techniques that require changes in the number or proportion of cell types, mRNA profiles and other cell isolation.

Thus, it was confirmed that the FRC identified through flow cytometry, as expected, forms a reticulum network throughout the T cell region (top panel). BEC expressed CD31 but did not express grapeplanin, and was present in the cortex mainly as a high endothelial vein distinguishable by its small size and cubic shape (top panel). LEC is characterized by co-expression of grapeplanin and CD31 and was lined with large lymphatic vessels in the hilum region of the water quality (center panel) and subcapsular (data not shown). Its gate region was also present in the large blood vessels (CD31 ⁺ grapes Plastic ^non-). In addition, MadCAM ⁺ reticulum cells lined under the capsule (bottom panel) and MadCAM staining were also present in the B cell region, as previously reported. In addition, it was confirmed that subcapsular LEC (where Lyve-1 expression was confirmed) also expressed MadCAM (bottom panel), forming a MadCAM-rich region of the lymph node. On the phenotype, MRC formed subspecies at the grapeplanin ⁺ CD31 ⁻ FRC gate by flow cytometry (FIG. 2B).

It has been hypothesized that the separation method of the present invention can be applied to separate human lymph node stroma, thereby constructing an in vitro experimental system. The human lymph node matrix structure is well documented histologically (Link et al., 2011), but no subspecies have been isolated in flow cytometry or other immunological experiments.

In the present invention, human lymph nodes of non-mesenteric origin of donor cadavers were obtained. To break down the lymph nodes, an increase in enzyme concentration was required (see method), but once digested, its substrate composition was quite similar to that of mice (compare Figures 2D and 2B). These results suggest that the murine lymph node stromal cell technique can be equally applied to human experiments.

Mesenteric lymph nodes contain less FRC than skin-draining lymph nodes.

To further verify the degradation, reproducibility was investigated by evaluating the difference in the proportion of lymph node stromal cell subspecies in mice of the same age. In addition, the matrix composition of skin-draining lymph nodes and mesenteric lymph nodes was also compared. These lymph nodes have well-established structural similarities and are well studied histologically, but their development occurs at different times and depends on different signaling pathways (Mebius, 2003). Additionally, mesenteric lymph nodes are continuously exposed to intestinal and gastric mucosa-derived antigens, while skin-draining lymph nodes are not.

Surprisingly, a similar number of cells were isolated from each skin-draining lymph node or mesenteric lymph node (Figure 3A), but the substrate composition was found to be significantly different (Figure 3B). FRCs growing throughout the T cell region are present at a higher frequency (FIG. 3B, FIG. 2B) and more in skin-draining lymph nodes than in mesenteric lymph nodes (FIG. 3C). LEC, BEC, DN and MRC were present in similar numbers at both sites (Figure 3C), showing FRC-specific differences.

It was confirmed that this difference was due to the rate distortion of the MRC subspecies (Katakai et al. 2008), and it was found to correspond to the FRC gate (FIG. 2B). 10-20% of the cells corresponding to the FRC gate expressed MadCAM in 5-week-old male mice, and the proportion was higher in the mesenteric lymph nodes than in the skin-draining lymph nodes (FIGS. 2B and 3D). However, the proportion of MRC was similar to LEC, but there was a significant difference between the two sites, but their total number was not (FIG. 3E). This suggests that the MRC number and FRC number are differentially regulated, while the MRC is numerically similar between the two sites, like other stromal subspecies, while the FRC is reduced in the mesenteric lymph nodes.

In some lymphoid organs, such as the thymus, the number of predominant stromal cell subspecies is amplified or reduced in parallel with T cell development. Rag ^-/- mice lacking T cells and B cells were tested to see if they apply to the FRC population. FRC occurs in Rag ^-/- lymph nodes, but it is not known whether they amplify and contract in response to lymphocyte count. This question is particularly relevant for infection, as the lymph nodes must rapidly amplify to contain proliferative lymphocytes in a short period of time.

Rag ^{− / −} mice had smaller lymph nodes (FIG. 3F), but had a normal number of FRCs compared to wild-type controls of the same age (FIG. 3G). However, the number of LECs is significantly reduced, suggesting that their numerical homeostasis is associated with the presence of mature lymphocytes. Rag ^-/- FRC did not differ from WT in terms of cell turnover rate (data not shown), and after injection of LPS into mice, the number of FRC was not amplified along with T cells and B cells (data not shown). . Thus, the number of FRCs remained completely independent of the mature lymphocytes in lymphocyte-reducing, steady-state and inflammatory conditions.

Surprisingly, it was confirmed that the MRC subset underestimated in Rag ^-/- mice (Fig. 3H-J). Histological methods have reported that MadCAM + MRC is present in Rag ^{− / −} mice (Katakai et al., 2008), but on the one hand, both the number of MadCAM + MRCs and the amount of MadCAM protein per MRC are significantly reduced (FIG. 2H- J). This also applies to MadCAM ⁺ LEC (Figure 3H-J), where upregulation of MadCAM expression in both cell types requires the presence of lymphocytes in the developmental stage, although protein self expression can be achieved in the absence thereof. Suggests that The ratio of MadCAM ⁺ LEC was similar in WT and Rag ^{− / −} (FIG. 3H), but a significant reduction in the total number of LECs (FIG. 3G) was interpreted as a reduction in the number of MadCAM ⁺ LECs (FIG. 3I).

Thickening and classification of lymph node stromal cell subspecies

After successfully dissolving the lymph nodes, the method was then modified to successfully sort and culture the stromal cells. Sorting of stromal cells can be technically difficult (see method), and in the present invention, MACS-based hematopoietic cell depletion was used to shorten sorting time and increase the purity obtained. This additional step minimized substrate loss (Figures 4A, B), and did not appreciably alter the substrate composition, resulting in no selective loss of substrate subspecies after MACS (Figure 4C) and significant purity achieved after sorting ( Figure 4D).

Using this method, FRCs from skin-draining lymph nodes and mesenteric lymph nodes were classified as part of the multicenter ImmGen Consortium. This involves transcryptom analysis of cells sorted under conditions of highly controlled and minimal variability, and the overall aim is to establish an open database to provide a comprehensive and cross-referenced map of the immunological genome. .

A probe expressing higher than an arbitrary threshold (average expression value> 120) in skin-draining lymph nodes or mesenteric lymph nodes by analyzing the expression of 25,194 probes in FRC derived from skin-draining lymph nodes and mesenteric lymph nodes We secured 11,162 types of lists. These probes were also filtered with a low (<0.5) coefficient of variation between replicas.

First, it was verified that FRCs at these various positions share the expression of previously published FRC-specific markers such as CD140a, CCL19 and IL-7 (data not shown), and then analyzed the differences in the data. . Comparison of skin-drained lymph node FRC vs. intestinal lymph node FRC, in a volcano plot showing fold-change and P-value, 130 probes in skin-drained lymph node derived FRC compared to mesenteric lymph node While significantly upregulated, the expression of 97 probes was found to be significantly higher in mesenteric lymph node FRC compared to skin-draining lymph nodes (> 2 fold change, P <0.05) (FIG. 5A). When these probes were mapped to native genes, these data corresponded to upregulation of 126 genes in skin-draining lymph node FRC (data not shown) and upregulation of 54 genes in mesenteric lymph node FRC (data shown). No).

We were interested in whether this list had evidence for activation of specific gene pathways, or whether the gene network works uniquely in both cell types as a clue to any site-specific specialization. Use the free online program DAVID (version 6.7; National Association of Allergic and Infectious Diseases), use network analysis tools (KEGG) to investigate known biological associations between these genes in the list, and then use statistical analysis tools Therefore, it was analyzed whether related genes appear together at a higher frequency than expected. The KEGG pathway mmu04060, which encompasses cytokine-cytokine receptor interactions, significantly enriched FRC in skin-draining lymph nodes compared to mesenteric lymph nodes (genes appear to be 4.8 times higher than accidentally predicted, Benza Mini-corrected P value = 0.0055). The list included 10 immunologically relevant genes that were up-regulated in skin-draining lymph nodes at least twice as large (FIG. 5B). Genes upregulated in mesenteric lymph nodes were not significantly enriched in the identifiable KEGG pathway, but genes involved in regulating fibroblast growth, genes related to intestinal microenvironment, such as genes involved in retinoic acid metabolism or MadCAM expression, endothelial cells Genes involved in cross-talk, extracellular matrix secretion and peripheral nerve outgrowth were included (Table 3).

The cultured FRC mimics in vivo function and supports leukocyte migration.

Next, the proliferative power of the lymph node stroma during the culture was examined. Successfully degraded lymph node cell suspension-derived substrate readily adhered to the tissue culture plate within 2 hours of inoculation and began to amplify as CD45 + leukocytes disappeared, and CD45 + leukocytes became less than 10% of the culture after 5 days (FIG. 6A). ). FRC and LCE proliferated easily in culture, but BEC and DN cells did not (FIG. 6A). FDC did not proliferate in culture (Figure 6B). Importantly, the low-trypsin, high EDTA recovery protocol of the present invention enables retention of important surface markers such as CD31 (Figure 6C) and CD140a (not shown; see Table 2). The present inventors confirmed that 5 x 10 ⁵ cells / cm ² containing about 5 x 10 ³ stromal cells usually becomes about 1.5-2 x 10 ⁴ stroma / cm ² after 5 days of culture. That is, this culture system can easily amplify and manipulate FRC, LEC and MRC.

In particular, FRC was highly biased during 2D (2D) culture (FIG. 6D), and strong F-actin stress fibers were identified (FIG. 6E). Since this does not mimic the in vivo form, as a tool for investigating their function when three-dimensional proliferation and connection are possible as such in vivo, Matrigel, an optimized mix of collagen I, and concentrated αMEM are used. Using, three-dimensional (3D) cultures of FRC and LEC were investigated. As a result, when cultured in 3D, the FRC unfolds to form a wide network (FIG. 6D, E), whereas the LEC prefers a flat surface to proliferate more successfully during 2D culture (FIG. 6D). Interestingly, the cell type did not proliferate to levels overconfluence in 3D conditions (data not shown), suggesting that there is an intrinsic regulation of cell density that does not occur in 2D culture. . In order to verify the biorelevance of this in vitro FRC network, two experiments were conducted. First, a small matrigel plug was inoculated with a large number of FRCs, and the ability to shrink the gel was monitored (FIG. 6F). FRC expresses α smooth muscle actin at high levels and has high contractility (Link et al., 2007). The inventors have found that FRC contracts the gel very effectively, and this contraction is prevented when Src tyrosine kinase inhibitor PP2 is added to the gel. Second, bone marrow-derived dendritic cells (DC) or spleen-derived lymphocytes are added to the FRC-containing gel and, using a live-imaging method, DC and lymphocytes are effective and preferentially FRC, as reported in vivo. It was confirmed that they traveled on the network (shown in FIG. 6G, full-type RAM movies are shown in Supplementary Movies 1 and 2).

Using the same method, human lymph node stromal cell subspecies were successfully cultured. Unlike murine cells, BRC and DN cells were cultured in vitro with LEC and FRC (Figure 6H).

In addition, these results indicate that the murine lymph node matrix can be easily separated for flow cytometry and sorting, cultured in the absence of white blood cells, and also, as reported in vivo, to form a wide 3D network in which leukocytes easily ride and move. Show. The data of the present invention suggest that human lymph node stroma can be treated similarly to experiments using the muline technique.

In addition, these data describe validation of a set of techniques applicable to studies of lymph node stromal cells in mice and humans, in vitro and in vitro.

Inhibitor cells derived from lymphoid tissue inhibit the proliferation of activated allogeneic splenocytes.

Unfractionated LSSCs were cultured with human spleen cells that were just isolated from unrelated donors. Splenocytes were labeled with a fluorescent dye, CFSE, which is diluted during cell division. T cells in this spleen cell population were activated using PHA and IL-2. 7 shows a histogram showing T cells identified through expression of the T cell antigen receptor. The ratio represents the ratio of splenocytes to stromal cells. T cells stimulated without stromal cell culture were more diluted in CFSE than cells cultured with LSSC, and LSSC inhibited T cell proliferation in a dose-dependent manner (FIG. 7).

LSSC inhibits the proliferation of activated heterologous T cells.

Unfractionated LSSC was incubated with mouse splenocytes that were isolated immediately. Splenocytes were labeled with a fluorescent dye, CFSE, which is diluted during cell division. T cells were activated using anti-CD3 and anti-CD28 antibodies. 8 shows a histogram showing T cells identified through expression of the T cell antigen receptor. The ratio represents the ratio of splenocytes to stromal cells. Blood-derived T cells stimulated without stromal cell culture were more diluted in CFSE than cells cultured with LSSC, and both the PDPN + and PDPN- LSSC subobject groups were able to inhibit T cells in the same way. LSSC is a potent inhibitor for heterologous responses (compared to the homologous inhibition shown in Figure 7).

LSSC inhibits T cell proliferation through a new mechanism.

In mice, lymph node-derived PDPN + stromal cells inhibit the proliferation of autologous T cells through mechanisms that decisively require T cell-derived IFNg and stromal cell-derived nitric oxide (Lukacs-Kornek et al. Nature Immunology, 2011). . The present inventors sought to investigate whether the LSSC of the present invention suppresses the T cell response using this same mechanism.

Unfractionated LSSCs were incubated with splenocytes just isolated from wild type or IFNg-/-mice. Splenocytes were labeled with CFSE, which is diluted upon cell division. T cells were then activated using anti-CD3 and anti-CD28 antibodies. 9A shows a histogram showing T cells identified through expression of the T cell antigen receptor. The ratio represents the ratio of splenocytes to stromal cells.

PDPN +, PDPN- and unfractionated LSSC were quickly isolated from unrelated donors and cultured with purified human blood-derived T cells. T cells were labeled with CFSE, which is diluted upon cell division. T cells were activated with PHA and IL-2. 9B shows a histogram showing T cells identified through expression of the T cell antigen receptor. The supernatant was collected to examine the presence of nitrite in the conversion product of nitric oxide.

This confirmed that LSSC inhibits IFNg-/-T cells as well as in wild type T cells. Neither the PDPN + or PDPN- LSSC subobject groups or the unfractionated LSSC produced detectable levels of nitric oxide / nitrite in the supernatant analysis. LSSC uses new IFNg and nitric oxide-independent mechanisms to inhibit T cell proliferation, and thus is significantly different from the mouse PDPN + lymph node matrix population.

The two main subpopulation groups of lymphoid trait-derived suppressor cells each inhibit the proliferation of activated allogeneic T cells.

In FIG. 10A, two subgroups of LSSC were purified based on the expression or deficiency of glycoprotein-36 (also known as PDPN). Both of these sub-subgroups were non-endothelial (CD31 negative) and non-hematopoietic (CD45 negative).

PDPN + LSSC, PDPN- LSSC, or unfractionated LSSC were incubated at 1: 5 ratio (LSSC: T cells) with freshly isolated and purified human blood-derived T cells from unrelated donors. T cells were labeled with CFSE, which is diluted upon cell division. T cells were activated with PHA and IL-2. 10B shows a histogram showing T cells identified through expression of the T cell antigen receptor. The ratio represents the ratio of splenocytes to stromal cells.

Blood-derived T cells stimulated without stromal cell culture were observed to have more CFSE dilution than cells cultured with LSSC. Both the PDPN + and PDPN- LSSC subobject groups are able to inhibit T cells equally.

LSSC stops division of pre-activated allogeneic T cells.

In some therapeutic uses of LSSC, individuals undergoing an immune response will require transfusion or transplantation. Thus, it was intended to confirm whether LSSC can inhibit pre-activated T cells.

PDPN + LSSC PDPN- LSSC, or unfractionated LSSC, was freshly isolated from unrelated donors and cultured with purified human blood-derived T cells. T cells were labeled with CFSE, which is diluted upon cell division. After activating T cells with PHA and IL-2, they were allowed to divide for 2 days, and then some T cells were inoculated onto LSSC to re-stimulate, or re-immunized to re-stimulate without LSSC, or re-immunized without re-stimulation. . 11 shows a histogram showing T cells identified through expression of the T cell antigen receptor. Ratios represent the ratio of LSSC to T cells.

T cells that were stimulated for 2 days and re-stimulated again with LSSC after re-immunization were confirmed to be sufficiently activated and cell division was stopped despite several cell divisions before re-immunization. , Not restimulated "shown with control). Both the PDPN + and PDPN- LSSC subobject groups are equally able to inhibit pre-activated allogeneic T cells.

LSSC significantly reduces the initial death of mice with severe graft versus host disease.

Allogeneic bone marrow transplantation was performed on the recipient mice (after split-dose lethal investigation, iv injection of total bone marrow). Severe GVHD was induced upon transplantation by co-injecting donor-derived splenocytes, including allergic T cells. Human LSSC 1 x 10 ⁶ were injected into the peritoneum at a certain time point after induction of GVHD, and control mice were provided with saline (vehicle). Viability was monitored.

Although all mice developed GVHD at about the same time, mice receiving LSSC were found to have significantly reduced mortality from GVHD at an early time point, and LSSC appears to lower the immune attack. This initial LSSC administration effect lasted for 3 weeks after injection, and the 3rd week is a period when protection is neutralized, so it seems that re-administration can be prescribed. When LSSC was administered to severe mice later (24 days), morbidity or mortality was not reversed.

As shown in FIG. 15, mouse lymph node stroma amplified in vitro lowers mortality due to acute sepsis shock in mice.

LSSC transcription features

Transcription characteristics of human LSSC were obtained using flow cytometry for surface markers and RT-PCR for secreted factors (FIG. 13A), and mRNA transcriptome was obtained using the Affymetrix HuGene ST1.0 gene array. (Fig. 13B).

A representative flow cytometry profile of LSSC (black line) (FIG. 13A) was compared to each negative staining control (gray), mesenchymal marker PDGFRa, tolerability related proteins PD-L1 and PD-L2, muscle-related proteins ITGA7 and lymph Expression of the characteristic proteins LTBR, IL7R and PDPN of the organ matrix was confirmed. PDPN and ITGA7 appeared to be expressed by subspecies of bulk LSSC. Substitute or gene names are indicated in parentheses. Through RT-PCR, it was confirmed that LSSC expresses the transcripts of chemokines CCL19 and CCL21 (positive control compared to LTbR).

Using gene arrays, the transcription of genes selected from three LSSC samples was analyzed, compared to published data for three human bone marrow mesenchymal stem cell samples (listing codes GM241199, GM241201, GM241203). According to the data (FIG. 13B), LSSC expressed PD-L2, LTbR, Itga7, Myh11 at high levels compared to BM-MSC. LSSC and MSC expressed CXCL12, Thy1, and IL7R at similar levels, and were negative for lineage-specific markers such as LYVE1, CD3G, PECAM1 (CD31), CD34, FCGR2B and PTPRC (CD45). It has been previously reported that MSC does not express PD-L2 and that this protein cannot be induced upon exposure to the inflammatory cytokine IFNg.

LSSC morphology

In the SCSC culture cultured in α-MEM supplemented with fetal bovine serum, a morphology similar to fibroblasts was observed (FIG. 14). It often exhibits long processes, lamellopodia, filopodia, ruffled leading edges, and focal adhesion. In addition, the LSSCs isolated from the lymph nodes and tonsils are similar in phenotype (FIG. 19).

FRC prevents sepsis in two different mouse models when administered at the time of treatment.

In both the aseptic sepsis model and the cecal ligation and perforation (CLP) sepsis model, FRC showed significant survival benefits (FIG. 16). 16E shows a reduction in the level of inflammatory cytokines in FRC-treated mouse blood. These inflammatory cytokines are generally associated with the innate immune system. In other words, FRC interacts with the innate immune system in sepsis.

FRC prevents colitis death.

Colonitis was induced by injecting purified native T cells into immunodeficient mice. Allogeneic FRC was administered 2 weeks after treatment (time of treatment) after the signs of disease appeared in the mouse and the weight began to decrease significantly. Compared to the brine-treated control, FRC was injected twice weekly from 2 weeks to 6 weeks, and survival was monitored. FRC had a significant effect on survival (FIG. 17).

Human LSSC secretes soluble cytokine factors, and cells isolated from lymph nodes and tonsils show similar levels to T cell suppression through soluble factors.

Cultured human LSSC has been shown to secrete a number of soluble cytokine factors (Figure 18). LSSCs isolated from lymph nodes and tonsils showed similar levels in T cell inhibition through soluble factors (FIG. 20).

Review

Techniques customarily used in the field of immunology and cell biology, such as preparing living single cell suspensions of renewable composition from primary tissues, cause problems when applied to lymph node stroma. As such, in vivo systems involving histology and pre-organ PCR, or lymphocyte-related readout are standard techniques in the field. They have made some important advances regarding the role of lymph node stromal cell subspecies in steady-state tolerance and immunity, but there are a number of questions that do not reveal direct biological and medical relevance. The present inventors thought that modern immunological and cellular biological techniques would be essential to addressing these questions, improving and adapting them to these rare and sophisticated cell types.

Highlighting these unmet needs, especially the DN substrate remains almost unidentified. Several groups currently report the appearance of non-hematopoietic stromal cell subspecies (which may or may not be allogeneic) (Link et al., 2007, Cohen et al., 2010, Fletcher et al., 2010), Although it constitutes> 10% of the lymph node stroma niche and possesses an Aire-expressing cell type (Cohen et al., 2010, Fletcher et al., 2010), it is almost unidentified. to be. Basic features such as shape, phylogeny, surface phenotype and function have not been published. This lack of information directly expresses the inherent difficulties in studying lymph node stromal cells.

In the DN cell research. One such problem is, you're dead or geotinde lymphocytes are poorly dye is being marked as negative or low in CD45, this same phenotype as the DN temperament in the stromal cells gated in (CD31 ^{- -} grape Playa Ninh) Is confirmed. This phenomenon greatly distorts the proportions of the true stroma subspecies, making it difficult to fix at a level similar to the most basic proportional composition of the lymph node stroma niche. Indeed, since the stroma is only 1-2% of the lymph node cells, non-surviving lymphocytes are generally more numerous than the stroma even in single cell suspensions with very high viability (survival> 95%). In the experiments of the present invention, staining of dead cells such as propidium iodide was included, so that cells that appeared to have low levels of CD45 after degradation were not inappropriately excluded.

For this reason, the inventors have found that the key to reproducible separation of substrates lies in the development of a low lethal enzymatic digestion protocol. By using a combination of dispase, DNase I and collagenase P, stromal cells were generally isolated with multiple viability> 95%. In addition, each stromal subspecies could be compared between individual mice and humans, and rare subspecies such as DN cells and MRCs could be successfully separated, which will be the subject of further research. No unique or differentially expressed markers and markers have been reported, so it is still impossible to identify the DN substrate histologically. However, using the classification strategy described herein, it is possible to perform the non-biased array-based approach necessary to identify the identity of these rare subspecies.

Likewise, the method is able to isolate four main populations of human lymph node stromal cells directly from primary tissue, opening the way for sophisticated clinical and pre-clinical studies. It is currently well established that mouse lymph node stromal cells express a number of clinically relevant auto-antigens and present them directly to T cells to induce immunological tolerance to them (Lee et al., 2007, Nichols et. al., 2007, Gardner et al., 2008, Magnusson et al., 2008, Cohen et al., 2010, Fletcher et al., 2010). It is possible that the same mechanism works in humans. Through experiments, it is unclear whether the constant transcription of the PTA in question or the retention of the peptide-MHC is the cause, but FRC and LEC possess the ability to present PTA to native T cells, and the interaction seen in vivo during culture It has been demonstrated to mimic action (Cohen et al., 2010, Fletcher et al., 2010). Using these techniques, we can solve these problems and many other important problems related to human lymph node biology.

Furthermore, the development of a 3D culture system for lymph nodes will enable the construction of highly controlled experimental systems that mimic interactions with the in vivo environment. In the present invention, it was confirmed that DC and lymphocytes migrate easily and preferably in vitro through the FRC network, as observed in vivo. These techniques are particularly applicable to research using human cells.

Slight differences were found in stromal cell subspecies between young C57Bl / 6 male mice of the same age. In testing whether cross-talk between lymphocytes and substrates is required to amplify specific substrate subspecies, FRC, LEC, BEC and DN substrates are present in Rag ^-/- mice in normal proportions, resulting in or It was confirmed that amplification shows that T cells or B cells are not required. MRC (MadCAM-1 ⁺ ) stromal subspecies has not been previously isolated for flow cytometry analysis, and it was confirmed in the present invention that MRC forms subspecies within the grapeplanin ⁺ CD31 ^- FRC gate. MRC exhibits similarity to FRC, expressing VCAM-1 and ICAM-1, releasing ECM components such as laminin, but additionally produces a large amount of CXCL13, and can be identified through expression of MadCAM-1 (Katakai et al., 2008).

Surprisingly, the number of MRCs was significantly reduced in Rag ^-/- mice. MadCAM protein levels per cell were also lowered. Histologically, MadCAM + MRC was visualized in skin-draining Rag ^-/- lymph nodes (Katakai et al., 2008), but numerical and quantitative flow cytometric analysis of MadCAM expression and MRC counts was previously impossible. These data indicate that lymphocytes are actually needed for normal MRC development. Interestingly, Rag ^-/- mouse-derived LEC also occurred in MadCAM + cells at a low rate, but a small amount of protein per cell was observed. It is still unclear whether MRC and MadCAM + LEC fail to amplify normally in Rag ^-/- mice or only to up-regulate MadCAM. Also, the role of MadCAM + LEC is unknown.

MRC shows the same phenotype as LTo cells (Katakai et al., 2008), since it has the lymph node downstream of lymphotoxin signaling and builds an NF-κB2 transcription program (Mebius, 2003, Coles et al., 2010) (Katakai et al., 2008). It has been established that can play a role in post-natal maintenance and regeneration of lymph node stroma (Katakai et al., 2008). As a result of the present invention, it appears that normal amplification and / or maturation of MadCAM + MRC is not required for normal amplification of FRC, BEC and DN substrates. However, additional functional experiments will be needed to confirm that MRC function (eg, chemokine expression) and MadCAM expression are necessarily linked.

What was unexpected was that the FRCs of the skin-draining and mesenteric lymph nodes differed significantly in terms of ratio, number and transcription profile. In the present invention, mesenteric lymph nodes with reduced expression of several important cytokines and chemokines: IL6, IL7, BAFF, CXCL9, CXCL10, IL1 receptor auxiliary protein (IL1RAP), activin receptor IIA, VEGFa, LIF, and cKIT-ligands In, it was confirmed that the number of FRCs was remarkably small. Statistically, route analysis showed that these genes were unlikely to be inadvertently clustered. You will be interested in determining whether these differences are the result of innate developmental differences or as a result of exposure to various inflammatory signals, such as antigenic exposure frequency or dose differences. Skin-draining lymph nodes and mesenteric lymph nodes are particularly exposed to various microenvironmental stimuli. As long as the skin boundaries are not destroyed, it can be said that in the present invention, FRCs present in the skin-draining lymph nodes are relatively unlikely to encounter large amounts of antigens of microbial origin. However, mesenteric FRC continues to be exposed to attack of pathogen-related molecular patterns through normal function of the intestine, and has an important steady-state role for Treg induction and oral tolerance (Hadis et al., 2011). It is reasonable to expect their daily factors to modify the expression profile as a result of indeterminate results.

Differences in occurrence between skin-draining lymph nodes and mesenteric lymph nodes have been reported: all of the mesenteric lymph nodes are present in Cxcl13-/-and Cxcr5-/-mice, but a number of skin-draining lymph nodes are missing or variably at specific locations Occurs (Ansel et al., 2000). Likewise, Ltb-/-mice develop mesenteric lymph nodes (albeit abnormally) but lack skin-draining lymph nodes (Koni et al., 1997). Since lymph node development is initiated at embryonic development, these results suggest that cytokines capable of complementing CXCL13 or LTβ are more available in mesenteric lymph nodes before birth than in skin-draining lymph nodes. Likewise, putative lymph node tissue cells are present in lower proportions in neonatal skin-draining lymph nodes than in mesenteric lymph nodes (Cupedo et al. J Imm 2004). Finally, after implanting the skin-draining lymph nodes into the mesentery, it was observed that MadCAM ^- BEC does not change to the mesenteric MadCAM + phenotype, suggesting that the difference is a developmental difference and not a result of exposure to the intestinal-derived antigen. (Ahrendt et al., 2008). In addition, in a recent study, interestingly, it was suggested that the substrate of the transplanted peripheral lymph node and mesenteric lymph node may have a difference in the inducing ability to induce oral tolerance, which is gradually replaced by donor cells after transplantation, carryover hematopoiesis. It is estimated that the cells have no influence (Buettner et al., 2011).

In the skin-draining lymph nodes of 6-week-old mice, it was confirmed that several cytokines with known roles in the lymph nodes are expressed at higher levels. In the FRC of skin-draining lymph nodes, IL6 was up-regulated 6-fold, a cytokine targeted for complex control. When produced early in the immune response after exposure to pro-inflammatory IL1, IL6 is a potent immunostimulatory factor, but can also exert anti-inflammatory functions through slowing TNFα and IL-1 signaling and up-regulation of IL10. IL10 was not expressed by the substrate (data not shown). Instead, the present invention confirmed> 3 fold up-regulation of IL1 receptor adjuvant protein (IL1Rap) in skin-draining lymph nodes, suggesting that skin-draining lymph node FRCs have the ability to increase IL6 production early in infection. do. Thus, during normal conditions, skin-drained lymph node-derived FRCs also expressed IL1R1, a high affinity binding partner of IL1Rap, at high levels, but did not express the decoy analog IL1-RN (Dinarello, 1996) (data not shown). . Based on these results, we have previously published that FRC responds to TLR3 signaling within hours (Fletcher et al., 2010), which means that FRC responds quickly to infection. In addition, as a means of stimulating phosphorylation of PDGF-receptors and cell contraction (Hu et al., 1998, Zampetaki et al., 2005), smooth muscle cells are elongated, thereby producing IL6 with anti-inflammatory functions (Pedersen, 2006) is also an important point. Likewise, in dermal myofibroblasts, IL6 regulates α smooth muscle actin to enhance contraction and wound closure (Gallucci et al., 2006). Like myofibroblasts, FRC are highly contractile cells expressing large amounts of α smooth muscle acti (Link et al., 2007, data not shown). IL6 also shares receptors with LIFs that are up-regulated in FRC derived from skin-draining lymph nodes. Like IL6, LIF is a means of inducing autocrine or paracrine muscle cell proliferation and is up-regulated in contractile muscle cells. IL6, IL1Rap and LIF are the three most upregulated genes by FRC derived from skin-draining lymph nodes in the identified network.

In addition to the direct relevance to lymphocyte function, the inventors have confirmed that IL7 and BAFF are up-regulated to more than 2-fold in skin-draining FRC. IL7 is a known product of FRC, and functions to maintain native T cells (Link et al., 2007). BAFF is a potent survival factor for B cells and a co-stimulatory active factor. In secondary lymphoid organs, it is known to be produced in non-hematopoietic FDC, but has not been previously reported in FRC. However, the expression of BAFF outside the lymph nodes by stromal cells has been well reported, but appears to cause chronic inflammation, and has not been associated with a steady-state matrix (Mackay et al., 2009). In the cell sorting method of the present invention, during the analysis of the transcription, no expression evidence of the Fcγ receptor was found, and <1% of lymph node stromal cells were found to be stained positively for FDC-M1 or CD35 (data not shown). Not included. FDCs are limited only to B cell follicles, while FRC extends around B cell follicles and sometimes to B cell follicles. However, both of these types of cells appear to arise from a common precursor (Mebius, 2003). This result is supported and expanded by a list referring to the similarities between these cell types.

In particular, the results of the present invention showed that CXCL9 and CXCL10 are expressed by FRC in skin-draining lymph nodes and mesenteric lymph nodes. Both of these chemokines transmit signals through CXCR3 expressed by activated T cells, B cells and NK cells, and their transcription is up-regulated by IFNγ, IFNα and other pro-inflammatory stimuli (Muller et al., 2010) . The role of CXCL10 in lymph nodes during steady state has not been identified, but in some infections, newly activated T cells also become Th1, a mechanism that causes a biased microbial-adaptive response, which is important for CXCL10 to retain Th1 lymphocytes in lymph nodes Plays a role (Yoneyama et al., 2002). However, to date, CXCL10 production has only been identified in mature DCs in lymphocytes (Yoneyama et al., 2002). These data show that CXCL10 mRNA is also expressed by steady-state FRC, but the function is undecided.

CXCL9 has not been thoroughly studied, but has been observed in cancer-related lymphoid tissue and drainage lymphoid-derived dendritic cells (Ohtani et al., 2009). No role has been reported in the regulation of steady state for cell migration in lymph nodes.

Interestingly, FRC derived from mesenteric lymph nodes and skin-draining lymph nodes expressed genes involved in signaling to endothelial cells. FRC is known to be the primary producer of VEGF present in skin-draining lymph nodes under steady-state conditions (Chyou et al., 2008). Surprisingly, it was confirmed that FRC derived from skin-draining lymph nodes expresses VEGFa at a higher level than mesenteric lymph nodes. Indeed, mesenteric lymph nodes up-regulated a relatively small list of genes compared to skin-draining lymph nodes, and the results did not map to functional pathways when performing a robust statistical correction for the multiple theory of the present invention. However, in the present invention, upregulation of known intestinal-related genes such as Nkx2-3 (Pabst et al., 2000) necessary for MadCAM expression and lymphocyte separation (lymphosyte segregation) in mesenteric lymph nodes was observed; In addition, up-regulation of Lrat, Tgm2, Meis1 and Pltp was also observed. These genes are involved in the retinoic acid metabolic cycle or are upregulated by retinoic acid (Matsuura et al., 1993, Cao et al., 2002, Galdones et al., 2006, Rebe et al., 2009), mesenteric lymphocytes Processed in to confer intestinal-specific regression phenotype on T cells (Iwata, 2009). In addition, the present invention confirmed the expression of several types of vasodilators (Vipr2, Itih4, Npr1) and vasoconstrictor (Agt), suggesting that FRC actively interacts with lymph node vessels. Likewise, upregulation of genes that induce neurite outgrowth (Efna5, gpr126, Tmem35 and Ngf) suggests a potential role in maintaining normal sympathetic nerves in the lymph nodes (Panuncio et al., 1999). Additional experiments will be needed to verify these results.

In short, the modification of expression of cytokines and chemokines in skin-draining lymph nodes provides a new picture of microenvironmental regulation by FRC. Expression of BAFF and CXCR3 suggests a cross-talk mechanism with the hematopoietic system that has not been previously reported. Likewise, upregulation of IL-6, IL1Rap and LIF, skin-draining lymph node FRCs may be more contractile, and / or divide more rapidly and vigorously than their mesenteric lymph node counterparts, and likewise increase VEGFα to increased levels. It suggests that by transcribing, it helps keep pace with endothelial cells.

However, this transfer phenomenon may not be the same in all FRCs. Lymph nodes contain a number of FRC microniches that have not been fully identified. The best studied for this is the cortical T cell region FRC (sometimes referred to as TRC), which produces IL-7, CCL19, and CCL21, which not only maintain the vascular system, but also for the migration of DC, T cells and B cells. Used as a scaffold. However, fibroblasts gp38 + CD31 ⁻ are present throughout the lymph nodes, with B cell follicles adjacent to the subcapsular region; Exists as a pericyte; It exists throughout the water quality department. It is still not possible to distinguish between these different FRC types, and if they differentially express the identified cytokines and chemokines, the difference between the skin-draining lymph nodes and the mesenteric lymph nodes may entail a difference in their proportions.

In sum, these data provide a comprehensive list of techniques designed for lymph node stromal cells.

Table 1 : Sample range of low pressure substrate classification parameters for 100 μm tip in FACSAria and FACSAria IIu (BD Biosciences). Independent experiment n = 4-8.

Classification parameters FACSAria FACSAria IIu Frequency 23.1-25.9 19.2-21.6 amplitude 24.0-45.6 8.6-13.5 Phase 0-3 0 Attenuation On Off Drop Delay 18.56-20.81 15.96-17.92 First Drop 189-295 200-243 Target Gap 6-10 10

Table 2: Enzyme sensitivity to surface markers used to characterize non-hematopoietic lymph node stromal cells. The cultured or isolated substrate was incubated with the enzyme for 10 minutes, and then surface protein expression was tested by flow cytometry. Data represent independent experiment n = 3.

Surface marker Collagenase P Dispase Trypsin CD31 + + - CD40 + + +/- CD44 + + + CD45 + + + CD80 + + - CD140a + + - ICAM-1 + + + Gp38 + + + Thy-1 + + +/- PD-L1 + + + Sca-1 + + + Lyve-1 + + + VCAM-1 + + + MadCAM + + +

+ Presence of surface markers (non-sensitive)

 -No surface marker (sensitivity)

+/- Surface marker is partially cut (partial sensitivity)

Table 3: Genes that are upregulated in mesenteric lymph node-derived FRC rather than skin-draining lymph node-derived FRC , grouped by functional similarity . Gene expression of classified FRCs derived from skin-draining lymph nodes or mesenteric lymph nodes was compared using microarray analysis. Genetic selection includes expression (average expression in SLN or MLN> 120), low coefficient of variation between replicas (<0.05), fold-change (more than 2-fold increase in mesenteric lymph node FRC compared to skin-drained FRC) and P- Based on the values (* <0.05; ** <0.01;student's T test). Genes were grouped based on literature keyword search results.

Functional group gene Multiple changes P-value Regulation of cell proliferation in fibroblasts







Wnt2b 8.13 ** Slit2 6.06 ** Sfrp4 3.13 ** Wfdc1 2.77 ** Mest 2.60 * Dhcr24 2.48 ** Fgfr3 2.39 ** Igfbp6 2.20 * Figf 2.17 ** Bmp2 2.12 ** Induction by intestinal-specific factors





Nkx2-3 5.75 ** Lrat 4.67 ** Tgm2 2.66 ** Enpp3 2.48 * Kcnn3 2.24 * Meis1 2.13 ** Pltp 2.07 ** Extracellular matrix or cell-cell junction interaction




Mfap5 3.38 * Hmcn2 2.69 ** Gpc3 2.34 ** Itih4 2.34 ** Cldn10 2.32 ** Col14a1 2.26 ** Loxl1 2.25 ** Cilp 2.01 * Cross-talk of endothelial or epithelial cells




Ptgs1 3.17 ** Vipr2 2.92 ** Smoc2 2.79 * Itih4 2.34 ** Npr1 2.33 * Figf 2.17 ** Agt 2.03 ** Neuron cross-talk
Efna5 2.58 ** Gpr126 2.22 ** Tmem35 2.04 ** Ngf 2.00 **

The invention is not limited to the details of the construction and arrangement of the set of components listed in the above description or illustrated in the drawings. The present invention is implemented in other embodiments, and may be implemented or implemented in various ways. In addition, the expressions and terminology used herein are for the purpose of description and should not be regarded as limiting. “Including,” “comprising,” or “having,” “containing,” “concomitant,” and variations thereof, are meant to encompass the items listed thereafter and their equivalents, as well as additional items.

Claims (35)

As a composition for suppressing the immune response,
The isolated fibroblast reticulocyte (FRC, fibroblastic reticular cell) contains an effective amount to suppress the immune response in the individual,
The FRC is passage-cultured, a composition for inhibiting an immune response. According to claim 1,
Wherein the FRC is an ex vivo expanded cell. According to claim 1,
Wherein the FRC is substantially free of non-FRC (non-FRC). According to claim 2,
The composition is administered to the individual in an amount of at least 100,000 FRC / kg. According to claim 3,
The composition is administered to the individual in an amount of 1,000,000 FRC / kg or more. According to claim 1,
The FRC is derived from one or more of lymph nodes, spleen, thymus, tonsils, adenoid and / or Peyer's patch. The method of claim 6,
The FRC is present on or within a two-dimensional or three-dimensional framework that is implanted into the subject. The method of claim 6,
The composition is administered to the subject by intravenous, intraperitoneal, intraarterial, subcutaneous or intramuscular injection, or by topical administration to a lesion, organ, organ capsule, fat or lymph node. The method according to any one of claims 1 to 8,
The subject is an individual suffering from an autoimmune disease or inflammatory disease. The method of claim 9,
The autoimmune disease or inflammatory disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, graft versus host disease and sepsis. The method according to any one of claims 1 to 8,
The composition wherein the FRC is autologous, allogeneic or heterologous to the subject. As a method of isolating fibroblast reticulocytes (FRC),
A method comprising the step of digesting a lymphatic tissue fragment using a combination and agitation of an enzyme mix and collecting and passage the FRC. The method of claim 12,
The breakdown of the lymphoid tissue fragment
(i) incubating the lymphoid tissue fragment with an enzyme mix;
(ii) stirring the tissue using a pipette, followed by incubating such that large fragments precipitate;
(iii) removing the supernatant and performing in a series of steps including repeating steps (i) and (ii) until all fragments are resolved,
The FRC is collected by combining all the supernatant fractions and then centrifuging to obtain a cell pellet. The method of claim 13,
The isolated FRC is cultured in a culture medium including a basic cell culture medium to which one or more of growth factors, serum, platelet cell blood, and antibiotics are added. The method of claim 14,
The isolated FRC is cultured to a density of 1-10,000 cells / cm ² . The method of claim 14,
The isolated FRC is cultured at 0.1-21% oxygen partial pressure. The method of claim 14,
The FRC is cultured until there is substantially no non-FRC present in the FRC. The method of claim 14,
The FRC is cultured until the number of stromal cells increases by at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200% or more. The method of claim 12,
The enzyme mix comprises a culture medium, dispase, collagenase and DNase I. The method of claim 12,
The method, wherein the FRC is derived from lymph nodes, spleen, thymus, tonsils, pharyngeal tonsils or Faer's plate. As a composition for suppressing the immune response,
Isolated fibroblast reticulocytes (FRC),
A composition for inhibiting an immune response, wherein the FRC is isolated using the method according to any one of claims 12 to 20. As a pharmaceutical composition,
It comprises a composition of isolated fibroblast reticulocytes (FRC),
The FRC is passage-cultured, pharmaceutical composition. The method of claim 22,
The FRC is a pharmaceutical composition that is isolated by treating a lymphatic tissue fragment using one or more of chemical, mechanical and electrical cell separation processes and then collecting the FRC. The method of claim 22,
The isolated FRC is amplified by culturing the collected cells until substantially no non-FRC is present in the FRC, the pharmaceutical composition. The method of claim 22,
The isolated FRC co-expresses CD140a and PD-L2, a pharmaceutical composition. The method of claim 22,
The isolated FRC co-expresses CD140a and LTBR, a pharmaceutical composition. The method of claim 22,
The isolated FRC co-expresses CD140a, PD-L2 and LTBR, pharmaceutical composition. The method of claim 22,
The isolated FRC expresses one or more other lymphoid markers selected from the group consisting of PD-L1, Thy-1, MADCAM-1, IL-7R or ITGA7. The method of claim 22,
The isolated FRC expresses IL-6, VEGF, or IL-6 and VEGF, pharmaceutical composition. The method of claim 22,
The cell is isolated from a species selected from the group consisting of humans, non-human primates, dogs, cats, horses, pigs, cows, and rodents. The method of claim 22,
The cell is a pharmaceutical composition that inhibits the proliferation of T cells in vitro. The method of claim 23,
The FRC is isolated from lymph nodes, spleen, thymus, tonsils, pharyngeal tonsils, or faceplates, pharmaceutical composition. delete delete delete

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